Abstract
In order to establish cells and organisms with predictable properties, synthetic biology makes use of controllable, synthetic genetic devices. These devices are used to replace or to interfere with natural pathways. Alternatively, they may be interlinked with endogenous pathways to create artificial networks of higher complexity. While these approaches have been already successful in prokaryotes and lower eukaryotes, the implementation of such synthetic cassettes in mammalian systems and even animals is still a major obstacle. This is mainly due to the lack of methods that reliably and efficiently transduce synthetic modules without compromising their regulation properties.To pave the way for implementation of synthetic regulation modules in mammalian systems we utilized lentiviral transduction of synthetic modules. A synthetic positive feedback loop, based on the Tetracycline regulation system was implemented in a lentiviral vector system and stably integrated in mammalian cells. This gene regulation circuit yields a bimodal expression response. Based on experimental data a mathematical model based on stochasticity was developed which matched and described the experimental findings. Modelling predicted a hysteretic expression responsewhich was verified experimentally. Thereby supporting the idea that the system is driven by stochasticity.The results presented here highlight that the combination of three independent tools/methodologies facilitate the reliable installation of synthetic gene circuits with predictable expression characteristics in mammalian cells and organisms.
Highlights
To date, most of the synthetic gene networks have been created for bacteria or for lower eukaryotes [1,2,3,4,5,6,7]
Bimodal expression from a lentiviral transduced positive feedback loop Efficient transduction and long-term stable expression of transgenes is achieved with the help of lentiviral transfer vectors both for basic research and gene therapeutic approaches [27]
A transcriptionally regulated gene circuit based on the Tetracycline(tet)system was implemented in the lentiviral vector
Summary
Most of the synthetic gene networks have been created for bacteria or for lower eukaryotes [1,2,3,4,5,6,7] They were successfully employed to investigate different regulatory networks within these biological systems. Stable integration of synthetic gene circuits remains a challenge and is up to now only achieved in immortalized cell lines which usually do not or only partially reflect relevant properties of the cells they have been derived of. This can be partially attributed to the genetic changes which are required to provide unlimited cell growth. To implement synthetic cassettes in primary cells novel tools have to be developed
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